In early testing, this hydrogel, developed by Johns Hopkins researchers, helped improve healing in third-degree burns. Photo by Will Kirk/HomewoodPhoto.jhu.edu

Johns Hopkins researchers have developed a jelly-like material and wound treatment method that, in early experiments on skin damaged by severe burns, appeared to regenerate healthy, scar-free tissue.

In the Dec. 12-16 online Early Edition of Proceedings of the National Academy of Sciences, the researchers reported their promising results from mouse tissue tests. The new treatment has not yet been tested on human patients. But the researchers say the procedure, which promotes the formation of new blood vessels and skin, including hair follicles, could lead to greatly improved healing for injured soldiers, home fire victims and other people with third-degree burns.

The treatment involved a simple wound dressing that included a specially designed hydrogel—a water-based, three-dimensional framework of polymers. This material was developed by researchers at Johns Hopkins’ Whiting School of Engineering, working with clinicians at the Johns Hopkins Bayview Medical Center Burn Center and the Department of Pathology at the university’s School of Medicine.

Third-degree burns typically destroy the top layers of skin down to the muscle. They require complex medical care and leave behind ugly scarring. But in the journal article, the Johns Hopkins team reported that their hydrogel method yielded better results. “This treatment promoted the development of new blood vessels and the regeneration of complex layers of skin, including hair follicles and the glands that produce skin oil,” said Sharon Gerecht, an assistant professor of chemical and biomolecular engineering who was principal investigator on the study.

Guoming Sun, left, a postdoctoral fellow, and Sharon Gerecht, an assistant professor of chemical and biomolecular engineering, helped develop a hydrogel that improved burn healing in early experiments. Photo by Will Kirk/HomewoodPhoto.jhu.edu

Gerecht said the hydrogel could form the basis of an inexpensive burn wound treatment that works better than currently available clinical therapies, adding that it would be easy to manufacture on a large scale. Gerecht suggested that because the hydrogel contains no drugs or biological components to make it work, the Food and Drug Administration would most likely classify it as a device. Further animal testing is planned before trials on human patients begin. But Gerecht said, “It could be approved for clinical use after just a few years of testing.”

John Harmon, a professor of surgery at the Johns Hopkins School of Medicine and director of surgical research at Bayview, described the mouse study results as “absolutely remarkable. We got complete skin regeneration, which never happens in typical burn wound treatment.”

If the treatment succeeds in human patients, it could address a serious form of injury. Harmon, a coauthor of the PNAS journal article, pointed out that 100,000 third-degree burns are treated in U. S. burn centers like Bayview every year. A burn wound dressing using the new hydrogel could have enormous potential for use in applications beyond common burns, including treatment of diabetic patients with foot ulcers, Harmon said.

Guoming Sun, Gerecht’s Maryland Stem Cell Research Postdoctoral Fellow and lead author on the paper, has been working with these hydrogels for the last three years, developing ways to improve the growth of blood vessels, a process called angiogenesis. “Our goal was to induce the growth of functional new blood vessels within the hydrogel to treat wounds and ischemic disease, which reduces blood flow to organs like the heart,” Sun said. “These tests on burn injuries just proved its potential.”

Gerecht says the hydrogel is constructed in such a way that it allows tissue regeneration and blood vessel formation to occur very quickly. “Inflammatory cells are able to easily penetrate and degrade the hydrogel, enabling blood vessels to fill in and support wound healing and the growth of new tissue,” she said. For burns, the faster this process occurs, Gerecht added, the less there is a chance for scarring.

Originally, her team intended to load the gel with stem cells and infuse it with growth factors to trigger and direct the tissue development. Instead, they tested the gel alone. “We were surprised to see such complete regeneration in the absence of any added biological signals,” Gerecht said.

Sun added, “Complete skin regeneration is desired for various wound injuries. With further fine-tuning of these kinds of biomaterial frameworks, we may restore normal skin structures for other injuries such as skin ulcers.”

Gerecht and Harmon say they don’t fully understand how the hydrogel dressing is working. After it is applied, the tissue progresses through the various stages of wound repair, Gerecht said. After 21 days, the gel has been harmlessly absorbed, and the tissue continues to return to the appearance of normal skin.

The hydrogel is mainly made of water with dissolved dextran—a polysaccharide (sugar molecule chains). “It also could be that the physical structure of the hydrogel guides the repair,” Gerecht said. Harmon speculates that the hydrogel may recruit circulating bone marrow stem cells in the bloodstream. Stem cells are special cells that can grow into practically any sort of tissue if provided with the right chemical cue. “It’s possible the gel is somehow signaling the stem cells to become new skin and blood vessels,” Harmon said.

Additional co-authors of the study included Charles Steenbergen, a professor in the Department of Pathology; Karen Fox-Talbot, a senior research specialist from the Johns Hopkins School of Medicine; and physician researchers Xianjie Zhang, Raul Sebastian and Maura Reinblatt from the Department of Surgery and Hendrix Burn and Wound Lab. From the Whiting School’s Department of Chemical and Biomolecular Engineering, other co-authors were doctoral students Yu-I (Tom) Shen and Laura Dickinson, who is a Johns Hopkins Institute for NanoBioTechnology (INBT) National Science Foundation IGERT fellow. Gerecht is an affiliated faculty member of INBT.

The work was funded in part by the Maryland Stem Cell Research Fund Exploratory Grant and Postdoctoral Fellowship and the National Institutes of Health.

The Johns Hopkins Technology Transfer staff has filed a provisional patent application to protect the intellectual property involved in this project.

Close-up of a cylindrically-shaped microfluidic device with two fluorescent solutions flowing through. Reproduced with permission from Nature Communications.

A leaf works something like a miniature laboratory. While the pores on the leaf surface allow it to channel nutrients in and waste products away from a plant, part of a leaf’s function also lies in its ability to curl and twist. Engineers use polymers to create their own mini-labs, devices called “labs-on-a-chip,” which have numerous applications in science, engineering and medicine. The typical flat, lab on a chip, or microfluidic device, resembles an etched microscopy cover slip with channels and grooves.

But what if you could get that flat lab-on-a-chip to self-assemble into a curve, mimicking the curl, twist or spiral of a leaf? Mustapha Jamal, a PhD student and IGERT fellow from Johns Hopkins Institute for NanoBioTechnology, has created a way to make that so.

Jamal is the lead author on “Differentially photo-crosslinked polymers enable self-assembling microfluidics,” published November 8, 2011 in Nature Communications. Along with principle investigator David Gracias, associate professor of Chemical and Biomolecular Engineering in the Whiting School of Engineering, and fellow graduate student Aasiyeh Zarafshar, Jamal has developed, for the first time, a method for creating three-dimensional lab-on-a-chip devices that can curl and twist.

The process involves shining ultraviolet (UV) light on a film of a substance called SU-8. Film areas closer to the light source become more heavily crosslinked than layers beneath, which on solvent conditioning creates a stress gradient.

Immersing the film in water causes the film to curl. Immersion in organic solvents like acetone causes the film to flatten. The curling and flattening can be reversed. The result, Jamal said, is the “self-assembly of intricate 3D devices that contain microfluidic channels.” This simple method, he added, can “program 2D polymeric (SU-8) films such that they spontaneously and reversibly curve into intricate 3D geometries including cylinders, cubes and corrugated sheets.”

Members of the Gracias lab have previously created curving and folding polymeric films consisting of two different materials. This new method achieves a stress gradient along the thickness of a single substance. “This provides considerable flexibility in the type and extent of curvature that can be created by varying the intensity and direction of exposure to UV light,” Gracias said.

Gracias explained that the method works with current protocols and materials for fabricating flat microfluidic devices. For example, one can design a 2D film with one type of lab-on-a-chip network, and then use their method to shape it into another geometry, also with microfluidic properties.

“Since our approach is compatible with planar lithography methods, we can also incorporate optical elements such as split ring resonators that have unique optical features. Alternatively, flexible electronic circuits could be incorporated and channels could be used to transport cooling fluids” Gracias said.

Tissue engineering is among the many important applications for 3D microfluidic devices, Gracias said. “Since many hydrogels can be photopolymerized, we can use the methodology of differential cross-linking to create stress gradients in these materials,” Gracias explained. “We plan to create biodegradable, vascularized tissue scaffolds using this approach.”

Peptide 'noodles' like this one can be made easily in a Petri dish. Photo by Martin Rietveld.

Everyone knows that uncooked spaghetti starts off aligned and ordered in the box but changes into a tangled mass of pasta once boiled in water. Imagine how difficult it would be to try to realign the spaghetti noodles.

Researchers in the chemistry laboratory of assistant professor J.D. Tovar at Johns Hopkins University have been making self-assembling nanowires made up of short peptide molecules with engineered electronic functions that have the potential for direct electronic interfacing with cells. The problem was, Tovar’s nanowire production method favored the fibers to form randomly jumbled networks, much like cooked spaghetti noodles on a plate. In that disordered arrangement, it would be difficult to control their alignment or allow them to interface with cells.

So Tovar and his PhD student, Brian Wall, turned to postdoctoral fellow Shuming Zhang from the lab of associate professor Hai-Quan Mao in the Department of Materials Science and Engineering in the Whiting School of Engineering. Wall, who is lead author on the paper recently published online in Advanced Materials, worked closely with Zhang and Stephen Diegelmann, one of Tovar’s former PhD students.

Together, the team created noodle-like arrays made up of self-assembling nanowires that all line up in the same direction to form thicker bundles with electrically conductive properties. Polarized optical microscopy and scanning electron microscopy were used to confirm that individual nanowires were nearly perfectly aligned within each noodle bundle.

With the help of PhD student Thomas Dawidczyk from the laboratory of the Howard Katz, professor and chair of Materials Science and Engineering, the team demonstrated that better alignment increases the fiber bundle’s ability to conduct electricity, Tovar said. The nanowire bundles “resemble a large electrical cable made up of individual wires,” Tovar added.

The long-term goal of the project is to use the nanowire noodles to forge electronic bridges with living cells or energy-relevant proteins. Tovar said improved nanowire alignment also is important to developing applications in tissue engineering. The aligned structure and peptide nature of these scaffold materials make it easier to functionalize these materials to guide cell growth.

The noodle fabrication technique involves the injection of a solution of peptide precursor molecules into another solution that facilitates the assembly of individual molecules into nanowires that are collectively aligned within the resulting noodle.

“It’s a method of making molecules into macro structures in one simple step,” Tovar said. The resulting noodle assemblies are up to a millimeter in diameter and several centimeters long. The next step, said Wall, will be finding ways to mass-produce and automate conductive noodle fabrication and develop them into functional devices.

Funding for this work was provided by a seed grant from Johns Hopkins Institute for NanoBioTechnology, the Department of Energy Office of Basic Energy Sciences, the National Science Foundation and the Maryland State Stem Cell Research Fund. The paper, “Aligned Macroscopic Domains of Optoelectronic Nanostructures Prepared via Shear-Flow Assembly of Peptide Hydrogels,” appears online prior to print in the journal Advanced Materials. Diegelmann was a Johns Hopkins Institute for NanoBioTechnology Integrative Graduate Education and Research Traineeship fellow. Additional authors on this research include William Wilson, formerly of the Johns Hopkins Integrated Imaging Center, who used confocal microscopy to explore the unusual electronic properties of the noodle assemblies.

The American Society for NanoMedicine (ASNM) will hold its third annual meeting November 9 -11 at the Universities at Shady Grove Conference Center in Gaithersburg, Md. This year ASNM has worked closely with the Cancer Imaging Program, National Cancer Institute, and National Institutes of Health to create a conference with a special focus on nano-enabeled cancer diagnostics and therapies, and the synergy of the combination of nano-improved imaging modalities and targeted delivery.

The program also focuses on updates on the newest Food and Drug Administration, nanotoxicity, nanoparticle characterization, nanoinformatics, nano-ontology, results of the latest translational research and clinical trials in nanomedicine, and funding initiatives. This year’s keynote speaker is Roger Tsien, 2008 Nobel Prize Laureate. Numerous other speakers and breakout sessions are planned for the three day event. Two speakers affiliated with Johns Hopkins include Justin Hanes and Dmitri Artemov. Hanes is a professor of nanomedicine in the department of ophthalmology at the Johns Hopkins School of Medicine. Artemov is an associate professor of radiology/magnetic resonance imaging research, also at the School of Medicine.

The deadline for the poster abstracts is October 1. The top four posters submitted by young (pre and post doctoral) investigators will be selected to give a short 10-minute (eight slides) oral presentation on November 11.

ASNM describes itself as a “a non-profit, open, democratic and transparent professional society…focus(ing) on cutting-edge research in nanomedicine and moving towards realizing the potential of nanomedicine for diagnosis, treatment, and prevention of disease.” More information about the ASNM can be found on the Society’s official website.

Students from Johns Hopkins Physical Sciences-Oncology Center (PSOC) and Center of Cancer Nanotechnology Excellence (CCNE) will hold their second mini-symposium of the year on October 10 at 9 a.m. in Hackerman Hall Auditorium. The symposia, scheduled each spring and fall on the Homewood campus, encourage an exchange of ideas between PhD students and postdoctoral fellows associated with these centers. The entire Hopkins community is invited to attend, and no RSVP is required.

Some of the talk titles include, from the department of Chemical and Biomolecular Engineering, “The Pulsing Motion of Breast Cancer Cell is Regulated by Surrounding Epithelial Cells” presented by Meng Horng Lee, a PSOC postdoctoral fellow in the Denis Wirtz lab; “Breast Tumor Extracellular Matrix Promotes Vasculogenesis” presented by Abigail Hielscher, a postdoctoral fellow in the Sharon Gerecht lab; and “Mucin 16 is a Functional Selectin Ligand on Pancreatic Cancer Cells” given by Jack Chen, a pre-doctoral fellow in the lab of Konstantinos Konstantopoulos. Additional speakers include postdoctoral fellow Pei-Hsun Wu, PhD, a from the Wirtz Lab and Koh Meng Aw Yong, a pre-doctoral student affiliated with Princeton University’s Physical Sciences-Oncology Center.

The purpose of these twice a year, student run mini-symposia is to facilitate communication among researchers working in laboratories studying the mechanistic aspects of cancer spread (i.e., those affiliated with the PSOC) and those working on novel means of using nanotechnology for cancer diagnosis or treatment (i.e., those associated with the CCNE). Anjil Giri coordinated the fall mini-symposium, a PSOC pre-doctoral fellow in the Wirtz lab , with Erbil Abaci, a PSOC pre-doctoral fellow with in the Gerecht lab. Visit the INBT website (inbt.jhu.edu) for further details, as additional speakers and talk titles will be announced.

To encourage promising high school students to pursue careers in academia and research, Johns Hopkins Institute for NanoBioTechnology and the Johns Hopkins School of Medicine welcome scholars from Baltimore’s Boys Hope Girls Hope (BHGH) to work in university laboratories. From June through August each summer for the past three years, high school students have worked alongside scientists in Johns Hopkins University laboratories producing raw data that supports the research goals of their mentors.

This summer, the university welcomed four BHGH scholars and, at the conclusion of the session, the scholars presented their findings to faculty, students, staff, and members of their families during a poster session held, August 12. The program also celebrated its first two high school graduates.

Matthew Green-Hill has been in the BHGH/INBT program for three summers. He graduated this spring from Archbishop Curley High School and was accepted to The College of William and Mary where he plans to study political science. He worked in the lab of assistant professor Sean Taverna in the department of pharmacology and molecular sciences. Along with his mentor PhD student Tonya Gilbert, Green-Hill presented “Cloning Yng1 to Identify Novel Histone Modification Binding Motifs that may affect Gene Expression” at the poster session.

Dwayne Thomas II worked in the cell biology laboratory of associate professor Douglas Robinson. He and his mentor, PhD student Hoku West-Foyle, conducted research that was presented in the poster “Dictyostelium discoideum myosin-ll, a modular motor.” Thomas has participated in the summer research program for two summers. He graduated from Loyola Blakefield in May and will attend Loyola University Maryland in the fall as a biology/pre-med major.

Working in the biological chemistry laboratory of professor Craig Montell, Durrell Igwe was mentored by postdoctoral fellow Marquis Walker and presented the poster “Reduced Immune Response in Drosophila Lysosomal Storage Disease Model.” This is also Igwe’s second year in the program, and he will graduate from Archbishop Curley High School in the spring of 2012.

One of the newest BHGH scholars is Charles Booth, who worked with postdoctoral fellow Yulia Artemenko in the cell biology lab of professor Peter Devreotes. He presented the poster “Analysis of the Functional Redundancy Between Dictyostelium KrsB and Its Mammalian Homolog Mstl.” Booth attends Calvert Hall and will be a junior this fall.

The BHGH program is geared toward students with academic potential but who lack the resources or stability to achieve their full potential. Some of those who have participated in the program may have at one time missed weeks of school in the past. Others have even been homeless. Students voluntarily apply to the nonprofit program to access services such as a stable home, tutoring, and counseling. Scholars have the opportunity to live together in an adult-supervised house in Baltimore and attend local private schools. Both boys and girls participate in the program and next year, Robinson said he hopes Hopkins will attract some of the young women interested in science and medicine to work in sponsored laboratories.

The Johns Hopkins University Imaging Initiative will host the first annual Imaging Conference, October 6, 2011 at the Turner Auditorium on the medical campus. The conference features afternoon lectures from various Hopkins faculty followed by a research poster session and happy hour. Anyone interested in imaging is welcome to attend.

Speakers include Elliot McVeigh, director of the Department of Biomedical Engineering; Elliot Fishman, MD, director of diagnostic imaging at body CT at Johns Hopkins Hospital; Jerry Prince, the William B. Kouwenhoven Professor of Electrical and Computer Engineering at the Whiting School of Engineering; Xingde Li, associate professor of biomedical engineering and head of the Laboratory of Biophotonics Imaging and Therapy at the Whiting School; Peter van Zijl, professor of radiology at the school of medicine and director of the F.M. Kirby Research Center for Functional Brain Imaging; and several others to be announced.

Three postdoctoral fellows from Johns Hopkins Institute for NanoBioTechnology will offer a one-hour crash course in how to get those research dollars; July 27, 11 a.m. Krieger 205. Free for Hopkins community.

Funding dollars make the research world go ‘round. Few know that better than postdoctoral fellows, who would be out of work without it. As part of Johns Hopkins Institute for NanoBioTechnology’s last professional development seminar of the summer, three INBT affiliated postdoctoral fellows will offer their sage advice on preparing winning research grants.

Biomedical engineers and clinicians at Johns Hopkins University have developed freeze-dried nanoparticles made of a shelf-stable polymer that only need the addition of water to activate their cancer-fighting gene therapy capabilities.

Principal investigator Jordan Green, assistant professor in the department of Biomedical Engineering at the Johns Hopkins School of Medicine, led the team that fabricated the polymer-based particles measuring 80 to 150 nanometers in diameter. Each particle, which is about the size of a virus, has the ability to carry a genetic cocktail designed to produce brain cancer cell-destroying molecules. After manufacture, the nanoparticles can be stored for up to 90 days before use. In principle, cancer therapies based on this technology could lead to a convenient commercial product that clinicians simply activate with water before injection into brain cancer tumor sites.

Because this method avoids the common, unpleasant side effects of traditional chemotherapy, “nanoparticle-based gene therapy has the potential to be both safer and more effective than conventional chemical therapies for the treatment of cancer,” Green said. But, he added current gene therapy nanoparticle preparations are just not practical for clinical use.

“A challenge in the field is that most non-viral gene therapy methods have very low efficacy. Another challenge with biodegradable nanoparticles, like the ones used here is that particle preparation typically takes multiple time-sensitive steps.” Green said. “Delay with formulation results in polymer degradation, and there can be variability between batches. Although this is a simple procedure for lab experiments, a clinician who wishes to use these particles during neurosurgery will face factors that would make the results unpredictable.”

In contrast, the nanoparticles developed by the Green lab are a freeze-dried, or “lyophilized,” formulation. “A clinician would simply add water, and it is ready to inject,” Green said. Green thinks this freeze-dried gene-delivery nanoparticle could be easily manufactured on a large scale.

Co-investigator Alfredo Quinones-Hinojosa, a Johns Hopkins Hospital clinician-scientist and associate professor in the departments of Neurosurgery and Oncology at the Johns Hopkins School of Medicine, said he could imagine particles based on this technology being used in conjunction with, and even instead of, brain surgery. “I envision that one day, as we understand the etiology and progression of brain cancer, we will be able to use these nanoparticles even before doing surgery,” Quinones said. “How nice would that be? Imagine avoiding brain surgery all together!”

Currently, patients with glioblastoma, or brain cancer, only have a median survival of about 14 months, Green said. “Methods other than the traditional chemotherapy drugs and radiation—or in combination with them—may improve prognosis,” he said.

Gene therapy approaches could also be personalized, Green said. “Because gene therapy can take advantage of many naturally-existing pathways and can be targeted to the cancer type of choice through nanoparticle design and transcriptional control, several levels of treatment specificity could be provided,” Green said.

The nanoparticles self-assemble from a polymer structural unit, so fabrication is fairly simple, said Green. Finding the right polymer to use, however, proved to be a challenge. Lead author Stephany Tzeng, a PhD student in biomedical engineering in Green’s lab screened an assortment of formulations from a “polymer library” before hitting on a winning combination.

“One challenge with a polymer library approach is that there are many polymers to be synthesized and nanoparticle formulations to be tested. Another challenge is designing the experiments to find out why the lead formulation works so well compared to other similar polymers and to commercially available reagents,” Green said.

Tzeng settled on a particular formulation of poly(beta-amino ester)s specifically attracted to glioblastoma (GB) cells and to brain tumor stem cells (BTSC), the cells responsible for tumor growth and spread. “Poly(beta-amino ester) nanoparticles are generally able to transfect many types of cells, but some are more specific to GBs and BTSCs,” Tzeng said.

The nanoparticles work like a virus, co-opting the cell’s own protein-making machinery, but in this case, to produce a reporter gene (used to delineate a tumor’s location) or new cancer fighting molecule. “It is possible that glioblastoma-derived cells, especially brain tumor stem cells, are more susceptible to our gene delivery approach because they divide much faster,” Tzeng added.

Not only are the particles convenient to use, the team discovered that dividing cells continued to make the new protein for as long as six weeks after application. “The gene expression peaked within a few days, which would correspond to a large initial dose of a therapeutic protein,” said Green. “The fact that gene expression can continue at a low level for a long time following injection could potentially cause a sustained, local delivery of the therapeutic protein without requiring subsequent injection or administration. The cells themselves would act as a ‘factory’ for the drug.”

Once the nanoparticles release their DNA cargo, Tzeng said the polymer quickly degrades in water, usually within days. “From there, we believe the degradation products are processed and excreted with other cellular waste products,” Tzeng said.

Members of the Green Lab are now working on identifying the intracellular mechanism responsible for facilitating cell-specific delivery. “We also plan to build additional levels of targeting into this system to make it even more specific. This includes modifying the nanoparticles with ligands to specifically bind to glioblastoma cells, making the DNA cargo able to be expressed only in GB cells, and using a DNA sequence whose product is only effective in GB cells.”

So far, the team has only successfully transfected brain tumor stem cells using these nanoparticles in a plastic dish. The next step is to test the particle in animal models.

“We hope to begin tests in vivo in the near future by implanting brain tumor stem cells into a mouse and injecting particles. We also hope to begin using functional genes that would kill cancer cells in addition to the fluorescent proteins that serve only as a marker,” Tzeng said.

Other authors who contributed to this work are Hugo Guerrero-Cázares, postdoctoral fellow in Neurosurgery and Oncology, and Joel Sunshine, an M.D.-Ph.D. candidate, and Elliott Martinez, an undergraduate leadership alliance summer student, both from Biomedical Engineering. Funding for this work came from the National Institutes of Health, Howard Hughes Medical Institute, the Robert Wood Johnson Foundation and a pilot-grant from Johns Hopkins Institute for NanoBioTechnology (INBT). Green is an affiliated faculty member of INBT. The research will be published in Issue #23 (August 2011) of the journal Biomaterials and is currently available online.